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WO2007026353A2 - Milieux de culture de cellules souches - Google Patents

Milieux de culture de cellules souches Download PDF

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Publication number
WO2007026353A2
WO2007026353A2 PCT/IL2006/000998 IL2006000998W WO2007026353A2 WO 2007026353 A2 WO2007026353 A2 WO 2007026353A2 IL 2006000998 W IL2006000998 W IL 2006000998W WO 2007026353 A2 WO2007026353 A2 WO 2007026353A2
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Prior art keywords
embryonic stem
stem cells
culture medium
cells
culture
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PCT/IL2006/000998
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English (en)
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WO2007026353A3 (fr
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Michal Amit
Joseph Itskovitz-Eldor
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Technion Research & Development Foundation Ltd.
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Priority to EP17197313.4A priority Critical patent/EP3354723B1/fr
Priority to AU2006286149A priority patent/AU2006286149B2/en
Priority to EP06766237A priority patent/EP1962719A4/fr
Priority to US11/991,077 priority patent/US8476070B2/en
Publication of WO2007026353A2 publication Critical patent/WO2007026353A2/fr
Publication of WO2007026353A3 publication Critical patent/WO2007026353A3/fr
Priority to US13/909,128 priority patent/US9404079B2/en
Priority to US15/209,776 priority patent/US10385312B2/en
Priority to US16/454,168 priority patent/US11512283B2/en
Priority to US17/971,677 priority patent/US20230060616A1/en

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    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to culture media, cell cultures and methods of culturing stem cells such as under defined and xeno-free culturing conditions.
  • hESCs Human Embryonic stem cells
  • FBS fetal bovine serum
  • MEFs inactivated mouse embryonic fibroblasts
  • MEF-conditioned medium or a MatrigelTM matrix which contains components from animal cells
  • the batch-specific variations may affect the quality of the culture.
  • human feeder-layer-based culture systems are xeno-free, they require the simultaneous growth of both feeder cell layers and hESCs, which limits the potential of large-scale culturing of hESCs.
  • culture systems based on feeder cells or conditioned medium are not well-defined and thus cannot be accurately repeated due to differences between the various lines of feeder-cells.
  • Xu C, et al. (Stem Cells, 2005, 23:315-23) developed a culture system based on a MatrigelTM matrix and a medium supplemented with serum replacementTM (SR), basic fibroblast growth factor (bFGF), with or without the addition of the Flt-3 ligand to the culture medium.
  • SR serum replacementTM
  • bFGF basic fibroblast growth factor
  • the background differentiation of the ESCs was 20 or 28 %, respectively, which is higher than observed for hESCs when cultured on MEFs.
  • Another culturing system based a MatrigelTM matrix and a medium supplemented with bFGF and Noggin, an antagonist of bone morphogenetic proteins (BMPs) 3 resulted in a background differentiation of 10 % (Xu RH., et al, 2005, Nat Methods. 2: 185-190).
  • BMPs bone morphogenetic proteins
  • both of these systems rely on MatrigelTM as a culturing matrix, their use for cell-based therapy is limited.
  • the present inventors have previously developed a culture system based on a fibronectin matrix and a medium supplemented with 20 % SR, transforming growth factor ⁇ l (TGFp 1 ) and bFGF (Amit et al, 2004).
  • one of the isolated hESC lines was reported to harbor a karyotype of 47,XXY after 4 months of continuous culturing and a second line, which was initially normal, converted to trisomy 12 between 4 and 7 months of culturing.
  • improvements of the feeder- free, xeno-free culturing systems of hESCs are highly needed.
  • Mouse ESCs can be continuously cultured without feeder layers provided that leukemia inhibitory factor (LIF) is added to the culture medium.
  • LIF leukemia inhibitory factor
  • hESCs accumulating data regarding hESCs suggest that LIF has no effect on preventing hESC differentiation (Thomson et al, 1998; Reubinof et al, 2000).
  • activation of key proteins of the LIF cellular pathway, such as signal transducer and activator of transcription 3 (STAT3) was found to be weak or absent in hESCs (Daheron et al, 2004; Humphrey et al, 2004; Sato et al, 2004).
  • the gpl30 receptor which is activated by ligands such as LIF, interleukin 6 (IL-6) and a chimera made of IL-6 and its soluble IL6 receptor (the IL6RIL6 chimera; Chebath et al, 1997), was shown to positively affect the mouse ESCs self-maintenance via STAT3 (Williams et al, 1988; Niwa et al, 1998; Smith -et al, 1988).
  • ligands such as LIF, interleukin 6 (IL-6) and a chimera made of IL-6 and its soluble IL6 receptor (the IL6RIL6 chimera; Chebath et al, 1997)
  • the IL6RIL6 chimera In hematopoietic stem cells, the IL6RIL6 chimera exhibited a much higher affinity for human gpl30 and was found to be more potent in increasing proliferation of progenitor cells than the mixture of IL-6 and the soluble IL6 receptor (Kollet et al, 1999). On the other hand, the IL6RIL6 chimera induced differentiation of ESC-derived oligodendrocyte precursors (Zhang PL., et al., 2006, MoL Cell Neurosci. 31: 387-398). In a recent study, Nichols et al., (1994) demonstrated that the IL6RIL6 chimera is capable of supporting mouse ESC culturing and derivation.
  • Daheron et al. (2004) showed that although the LIFR ⁇ and the signaling subunit gpl30 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs, human LIF is unable to maintain the pluripotent state of hESCs.
  • Humphrey et al. (2004) found that hESCs rapidly differentiate when cultured in a medium containing members of the IL-6 family of cytokines such as LIF, IL-6 or a complex of the soluble IL-6 receptor and IL-6 (the "hyper-IL-6") and concluded that maintenance of pluropotency in human ESCs is STAT independent.
  • a culture medium being serum-free, xeno-free, feeder-free and protein carrier-free and capable of maintaining stem cells in an undifferentiated state.
  • a cell culture comprising a stem cell and a culture medium, said culture medium being serum-free, xeno-free, feeder-free and protein carrier-free and capable of maintaining said stem cells in an undifferentiated state.
  • a method of expanding and maintaining stem cells in an undifferentiated state comprising culturing the stem cells in a culture medium being serum-free, xeno-free, feeder-free and protein carrier-free and capable of maintaining the stem cells in an undifferentiated state, thereby expanding and maintaining the stem cells in the undifferentiated state.
  • a method of deriving an embryonic stem cell line comprising: (a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing said embryonic stem cell in a culture medium being serum-free, xeno-free, feeder-free and protein carrier-free and capable of maintaining said embryonic stem cell in an undifferentiated state; thereby deriving the embryonic stem cell line.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium being serum- free, xeno-free, feeder-free and protein carrier-free and capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage-specific cells from the embryonic stem cells.
  • a method of generating embryoid bodies from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium being serum- free, xeno-free, feeder-free and protein carrier-free and capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said embryonic stem cells to embryoid bodies; thereby generating the embryoid bodies from the embryonic stem cells.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium being serum-free, xeno-free, feeder-free and protein carrier-free and capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated embryonic stem cells to embryoid bodies; and (c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage- specific cells from the embryonic stem cells.
  • a culture medium comprising a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, wherein the culture medium is capable of maintaining stem cells in an undifferentiated state.
  • a cell culture comprising a stem cell and a culture medium, said culture medium comprising a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, wherein said culture medium is capable of maintaining said stem cell in an undifferentiated state.
  • a method of expanding and maintaining stem cells in an undifferentiated state comprising culturing the stem cells in a culture medium which comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, said culture medium is capable of maintaining the stem cells in an undifferentiated state, thereby expanding and maintaining the stem cells in the undifferentiated state.
  • a method of deriving an embryonic stem cell line comprising: (a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing said embryonic stem cell in a culture medium which comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, thereby deriving the embryonic stem cell line.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage-specific cells from the embryonic stem cells.
  • a method of generating embryoid bodies from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said embryonic stem cells to embryoid bodies; thereby generating the embryoid bodies from the embryonic stem cells.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated embryonic stem cells to embryoid bodies; and (c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage-specific cells from the embryonic stem cells.
  • a culture medium comprising an IL6RIL6 chimera, wherein the culture medium is capable of maintaining human embryonic stem cells in an undifferentiated state.
  • a cell culture comprising a human embryonic stem cell and a culture medium, said culture medium comprising an IL6RIL6 chimera, wherein said culture medium is capable of maintaining said human embryonic stem cell in an undifferentiated state.
  • a method of expanding and maintaining human embryonic stem cells in an undifferentiated state comprising culturing the human embryonic stem cells in a culture medium which comprises an IL6RIL6 chimera, said culture medium is capable of maintaining the human embryonic stem cells in an undifferentiated state, thereby expanding and maintaining the embryonic stem cells in the undifferentiated state.
  • a method of deriving a human embryonic stem cell line comprising: (a) obtaining a human embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing said human embryonic stem cell in a culture medium which comprises an IL6RIL6 chimera, said culture medium is capable of maintaining the human embryonic stem cells in an undifferentiated state, thereby deriving the embryonic stem cell line.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises an IL6RIL6 chimera, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage- specific cells from the embryonic stem cells.
  • a method of generating embryoid bodies from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises an IL6RIL6 chimera, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said embryonic stem cells to embryoid bodies; thereby generating the embryoid bodies from the embryonic stem cells.
  • a method of generating lineage-specific cells from embryonic stem cells comprising: (a) culturing the embryonic stem cells in a culture medium which comprises an IL6RIL6 chimera, said culture medium is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; (b) subjecting said expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated embryonic stem cells to embryoid bodies; and (c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage- specific cells from the embryonic stem cells.
  • the embryonic stem cells are human embryonic stem cells.
  • the culture medium comprising a TGF ⁇ isoform.
  • the culture medium being serum replacement-free. According to still further features in the described preferred embodiments the culture medium comprising IL6RIL6 chimera.
  • the culture medium comprising IL6RIL6 chimera and whereas said stem cells are human embryonic stem cells. According to still further features in the described preferred embodiments the culture medium comprising IL6RIL6 chimera and whereas said embryonic stem cells are human embryonic stem cells.
  • the stem cells are embryonic stem cells.
  • the culture medium is capable of expanding said stem cells in an undifferentiated state.
  • the protein carrier is albumin.
  • culturing is effected in suspension.
  • the suspension is devoid of substrate adherence.
  • culturing is effected on a feeder-layer free matrix.
  • the feeder-layer free matrix is a fibronectin matrix.
  • the culturing is effected on feeder cells.
  • the culture medium is xeno-free.
  • culturing is effected in xeno-free culturing conditions.
  • the TGF ⁇ isoform is a TGF ⁇ isoform 1 (TGFp 1 ).
  • the TGF ⁇ isoform is a TGF ⁇ isoform 3 (TGFp 3 ). According to still further features in the described preferred embodiments the
  • TGF ⁇ i is provided at a concentration of at least 0.06 ng/ml.
  • the TGF ⁇ i is provided at a concentration of 0.12 ng/ml.
  • the TGF ⁇ 3 is provided at a concentration of at least 0.5 ng/ml.
  • the TGF ⁇ 3 is provided at a concentration of 2 ng/ml.
  • the culture medium comprises basic fibroblast growth factor (bFGF).
  • bFGF basic fibroblast growth factor
  • the bFGF is provided at a concentration of at least 2 ng/ml.
  • the bFGF is provided at a concentration of at least 4 ng/ml.
  • the IL6RIL6 chimera is provided at a concentration of at least 25 ng/ml.
  • the culture medium comprises serum or serum replacement.
  • the culture medium is devoid of serum or serum replacement. According to still further features in the described preferred embodiments the serum or serum replacement is provided at a concentration of at least 10 %.
  • the method further comprising isolating lineage specific cells following step (b).
  • isolating lineage specific cells is effected by a mechanical separation of cells, tissues and/or tissue-like structures contained within said embryoid bodies. According to still further features in the described preferred embodiments isolating lineage specific cells is effected by subjecting said embryoid bodies to differentiation factors to thereby induce differentiation of said embryoid bodies into lineage specific differentiated cells. According to still further features in the described preferred embodiments the embryonic stem cell is a human embryonic stem cell.
  • the embryonic stem cell is a primate embryonic stem cell.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing a well-defined, xeno-free culture media which comprise a TGF ⁇ isoform or the IL6RIL6 chimera, which are capable of mainataining stem cells in an undifferentiated state.
  • FIGs. la-f are photomicrographs depicting examples of the morphology of undifferentiated ESC colonies and ESC single cells grown in various feeder-free culture systems.
  • Figure Ic Undifferentiated 1-3 colony cultured on a mouse laminin-based feeder layer free culture system for 7 passages in the presence of a culture medium supplemented with 100 ng/ml of the IL6RIL6 chimera. Note the absence of "auto-feeders”.
  • FIG. 2 depicts a normal karyotype of an exemplary hESC.
  • the karyotype was examined after 7 passages of culturing in the presence of a culture medium containing 300 ng/ml of the IL6RIL6 chimera on a fibronectin feeder-free culture system. Note the presence of a normal 46,XX karyotype. Normal karyotype was also detected when the hESCs were cultured for 7 passages in the presence of a culture medium containing 100 ng/ml of the IL6RIL6 chimera on a fibronectin feeder-free culture system (not shown). Repeated test at passage 23 was found to be normal (not shown). 40 metaphases were examined from each sample.
  • FIGs. 3a-c are photomicrographs depicting immunofluorescence staining of undifferentiated colonies stained with surface markers specific to the hESC undifferentiated stage.
  • FIGs. 4a-l are RT-PCR analyses depicting the expression of representative genes of the undifferentiated stage and of the three embryonic germ layers in hESCs grown on human fibronectin feeder free culture system or in embryoid bodies (EBs) derived therefrom.
  • Lane 2 Cell line 1-3 cultured for 12 passages in 300 ng/ml of the IL6RIL6 chimera.
  • Lane 3 10-day-old EBs derived from 1-3 cells which were cultured for 10 passages in CMlOO.
  • Lane 4 - EBs derived from 1-3 cells which were cultured for 10 passages in 300 ng/ml of the IL6RIL6 chimera.
  • FIGs. 5a-d are photomicrographs depicting the morphology of undifferentiated hES colonies and hES single cells cultured in various culture systems in the presence of the TGF ⁇ -containing culture media.
  • Figure 5d - 14 hESCs cultured for 11 passages on a human fibronectin matrix with the D2 medium Note the undifferentiated morphology after prolonged culturing with the unique TGF ⁇ - containing media types.
  • Magnifications are X 15 for Figures 5a-d.
  • FIGs. 6a-c are photomicrographs depicting undifferentiated colonies stained with surface markers specific to the hESC undifferentiated stage. 14 hESCs cultured for 36 passages on a MatrigelTM matrix with the medium Dl and stained with TRA-I- 60 ( Figure 6a), SSEA4 ( Figure 6b) and TRA- 1-81 ( Figure 6c); Magnifications are X
  • FIGs. 7a-b are photomicrographs depicting the derivation of a new hESC line under xeno-free conditions on foreskin fibroblasts using the HAl 6 medium.
  • FIGs. 8a-c are photomicrograph depicting immunostaining of hESCs cultured for 3 passages in suspension in the presence of the D2 medium. Shown are immunostaining analyses of Oct4 ( Figure 8a), TRA- 1-60 ( Figure 8b) and TRA- 1-81 ( Figure 8c); Magnifications are X 63 for Figures 8a-c.
  • FIGs. 9a-g are photomicrographs depicting histological sections and morphology of suspended hESCs culture.
  • Figures 9d-e - 14 hESCs were cultured for 16 passages in suspension in the presence of the CMlOOF medium and were then re-cultured on MEFs. Shown is the morphology of colonies after re-culturing on MEFs. Note the typical undifferentiated morphology of the hESCs.
  • Figures 9f-g - 14 hESCs were cultured for 7 passages in suspension in the presence of the HA 19 medium ( Figure 9f) or for 10 passages in the presence of the CMlOOF medium (Figure 9g); Magnifications are X 20 for Figure 9a and X 15 for Figures 9b-g.
  • FIGs. 10a-d are RT-PCR analyses depicting the expression of representative genes of the undifferentiated state of hESCs cultured in suspension in the presence of the HACMlOO, CMlOOF or the HA19 medium.
  • Lane 2 - 1-4 hESCs cultured for 1 passage in suspension in the presence of the CMlOOF medium IL6RIL6 and serum replacement-containing medium).
  • Figure 10a - Oct4; Figure 10b - Rexl; Figure 10c — Sox2; Figure 1Od - Nanog; RT mix were tested and found negative for all tested genes. All samples were tested for ⁇ -actin and were found evenly positive.
  • the present invention is of well-defined, xeno-free culture media which comprise a TGF ⁇ isoform or the IL6RIL6 chimera, which are capable of mainataining stem cells in an undifferentiated state.
  • the present invention is of cell cultures comprising the culture media and the stem cells and of methods of expanding and deriving embryonic stem cells in such well-defined, xeno-free culture media.
  • the present invention is of methods of differentiating ESCs or EBs formed therefrom for the generation of lineage specific cells.
  • the present invention can be used to generate highly reproducible, xeno-free cultures of hESCs which can be used for both cell-based therapy, pharmaceutical screening, identification of drug targets and cell-based compound delivery.
  • hESCs Human embryonic stem cells
  • hESCs Human embryonic stem cells
  • hESCs are proliferative, undifferentiated, stem cells capable of differentiating into cells of all three embryonic germ layers.
  • hESCs are employed as a research model for early human development and hold promise for various applications including cell-based therapy, pharmaceutical screening, identification of drug targets and cell-based compound delivery which require almost indefinite amounts of proliferating, yet pluripotent hESCs.
  • hESCs For use in human therapy and for the production of large amounts of hESCs, hESCs need to be cultured in complete animal-free (xeno-free), well-defined culture systems, where exposure to animal (e.g., retroviruses) or human pathogens and batch-dependent variations are avoided.
  • Attempts to achieve defined and/or xeno-free hESCs culture systems include the development of a culture system which is based on a fibronectin matrix and a medium supplemented with 20 % SR, transforming growth factor P 1 (TGFp 1 ) and bFGF [Amit et al, 2004] or the culture system based on a matrix consisted of the combination of human collagen IV, fibronectin, laminin and vitronectin and a medium supplemented with human serum albumin, bFGF and TGFp 1 (Ludwig et al, 2006).
  • TGFp 1 transforming growth factor P 1
  • bFGF transforming growth factor P 1
  • LIF leukemia inhibitory factor
  • a recombinant polypeptide which includes the IL-6 and the soluble IL-6 receptor (the IL6RIL6 chimera, Chebath J, et al., 1997), was shown to support mouse ESC culturing and derivation (Nichols et al., 1994).
  • IL6RIL6 chimera the IL6RIL6 chimera
  • hlL- the soluble hIL-6R and hlL-
  • the common knowledge is that in contrast to mouse ESCs which can be maintained in the undifferentiated state in the presence of activators of the gpl30 receptor, culturing of human ESCs in the presence of LIF, IL6 or the "hyper IL6" results in differentiation of the hESCs. While reducing the present invention to practice, the present inventors have uncovered through laborious experimentations, a well-defined, xeno-free, serum or serum replacement-free and protein carrier-free culture medium which comprises a TGF ⁇ isoform and which can be used for maintaining hESCs in a pluripotent and undifferentiated state.
  • hESCs cultured on a human fibronectin matrix in the presence of a well-defined TGF ⁇ -containing culture medium which is devoid of serum, serum replacement and a protein carrier (e.g., albumin) exhibited the morphology of undifferentiated ESC colonies and ESC single cells ( Figures 5a-d) and expressed typical hESC surface markers specific to the undifferentiated state ( Figures 6a-c).
  • a protein carrier e.g., albumin
  • the present inventors have uncovered that the new TGF ⁇ -containing culture medium is suitable for the successful derivation of new hESC lines ( Figures 7a-b, Example 3 of the Examples section which follows) on a complete xeno-free culture system.
  • the culture medium comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier, wherein the culture medium is capable of maintaining stem cells in an undifferentiated state.
  • culture medium refers to a solid or a liquid substance used to support the growth of stem cells and maintain them in an undifferentiated state.
  • culture medium refers to a liquid substance capable of maintaining the stem cells in an undifferentiated state.
  • the culture medium used by the present invention can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all ⁇ of which are needed for cell proliferation and are capable of maintaining the stem cells in an undifferentiated state.
  • a culture medium according to this aspect of the present invention can be a synthetic tissue culture medium such as Ko-DMEM (Gibco-Invitrogen Corporation products, Grand Island, NY, USA), DMEM/F12 (Biological Industries, Biet Haemek, Israel), Mab ADCB medium (HyClone, Utah, USA) or DMEM/F12 (Biological Industries, Biet Haemek, Israel) supplemented with the necessary additives as is further described hereinunder.
  • all ingredients included in the culture medium of the present invention are substantially pure, with a tissue culture grade.
  • stem cells refers to cells which are capable of differentiating into other cell types or remaining in an undifferentiated state.
  • stem cells encompasses embryonic stem cells (ESCs), adult stem cells and hematopoietic stem cells.
  • embryonic stem cells refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state.
  • embryonic stem cells may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation (i.e., a pre- implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre-gastrulation stage blastocyst (see WO2006/040763 to the present inventors] and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.
  • gestation e.g., blastocyst
  • EBCs extended blastocyst cells
  • EG embryonic germ
  • tissue stem cells include stem cells derived from any adult or fetal tissue such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine and bone marrow.
  • Hematopoietic stem cells which may also referred to as adult tissue stem cells, include stem cells obtained from blood or bone marrow tissue of an individual at any age or from cord blood of a newborn individual.
  • Preferred stem cells according to this aspect of the present invention are embryonic stem cells, preferably of a human or primate (e.g., monkey) origin.
  • the embryonic stem cells of the present invention can be obtained using well- known cell-culture methods.
  • human embryonic stem cells can be isolated from human blastocysts.
  • Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos.
  • IVF in vitro fertilized
  • a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting.
  • ICM inner cell mass
  • the ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol.
  • ES cells can also be used with this aspect of the present invention.
  • Human ES cells can be purchased from the NIH human embryonic stem cells registry (http://escr.nih.gov).
  • Non-limiting examples of commercially available embryonic stem cell lines are BGOl, BG02, BG03, BG04, CY12, CY30, CY92, CYlO, TE03 and TE32.
  • EBCs Extended blastocyst cells
  • EBCs can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation.
  • the zona pellucida Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode's acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass.
  • the blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.
  • EG cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts.
  • the genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation.
  • the EG cells are then grown in tissue culture flasks with the appropriate medium.
  • the cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.
  • Shamblott et al. [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No.
  • Fetal stem cells can be isolated using various methods known in the art such as those disclosed by Eventov-Friedman S, et al., PLoS Med. 2006, 3: e215; Eventov-Friedman S, et al., Proc Natl Acad Sci U S A. 2005, 102: 2928-33; Dekel B, et al., 2003, Nat Med. 9: 53-60; and Dekel B, et al., 2002, J. Am. Soc. Nephrol.
  • Hematopoietic stem cells can be isolated using various methods known in the arts such as those disclosed by "Handbook of Stem Cells” edit by Robert Lanze, Elsevier Academic Press, 2004, Chapter 54, pp609-614, isolation and characterization of hematopoietic stem cells, by Gerald J Spangrude and William B Stayton.
  • stem cells in an undifferentiated state are of a distinct morphology, which is clearly distinguishable by the skilled in the art from that of differentiated cells of embryo or adult origin.
  • undifferentiated stem cells typically have high nuclear/cytoplasmic ratios, prominent nucleoli and compact colony formation with poorly discernable cell junctions. Additional features of the undifferentiated state of the stem cells are further described hereinunder.
  • the culture medium according to this aspect of the present invention comprises a TGF ⁇ isoform and being devoid of serum, serum replacement and protein carrier.
  • TGF ⁇ isoform refers to any isoform of the transforming growth factor beta ( ⁇ ) including TGF ⁇ t (e.g., homo sapiens TGF ⁇ l3 GenBank Accession No. NP 000651), TGF ⁇ 2 (e.g., homo sapiens TGF ⁇ 2 , GenBank Accession No. NP_003229) and TGF ⁇ 3 (e.g., homo sapiens TGF ⁇ 3 , GenBank Accession No. NP_003230) which function through the same receptor signaling system in the control of proliferation, differentiation, and other functions in many cell types.
  • TGF ⁇ acts in inducing transformation and also acts as a negative autocrine growth factor.
  • the TGF ⁇ isoform which is included in the culture medium of the present invention is TGF ⁇ t or TGF ⁇ 3 .
  • TGF ⁇ isoforms can be obtained from various commercial sources such as R&D Systems Minneapolis MN, USA.
  • TGF ⁇ t e.g., the Dl medium which contains 0.12 ng/ml TGF ⁇ t
  • TGF ⁇ 3 e.g., the D2 medium, the HAl 6 medium or the HAl 9 medium which contain 2 ng/ml TGF ⁇ 3
  • TGF ⁇ t which is included in the culture medium of this aspect of the present invention is provided at a concentration of at least 0.06 ng/ml, more preferably, at least 0.07 ng/ml, more preferably, at least 0.08 ng/ml, more preferably, at least 0.09 ng/ml, more preferably, at least 0.1 ng/ml, more preferably, at least 0.11 ng/ml, even more preferably, at least 0.12 ng/ml.
  • TGF ⁇ 3 which is included in the culture medium of this aspect of the present invention is provided at a concentration of at least 0.5 ng/ml, more preferably, at least 0.6 ng/ml, more preferably, at least 0.8 ng/ml, more preferably, at least 0.9 ng/ml, more preferably, at least 1 ng/ml, more preferably, at least 1.2 ng/ml, more preferably, at least 1.4 ng/ml, more preferably, at least 1.6 ng/ml, more preferably, at least 1.8 ng/ml, even more preferably, at least 2 ng/ml.
  • the TGF ⁇ -containing culture medium of this aspect of the present invention further includes other growth factors such as basic fibroblast growth factor (bFGF).
  • bFGF basic fibroblast growth factor
  • tissue culture ingredients such as Invitrogen Corporation products, Grand Island NY, USA.
  • a DMEM/F12 - based culture medium e.g., HA16 or HA19 medium which includes TGF ⁇ 3 and 4 ng/ml bFGF
  • Mab ADCB medium - based culture medium e.g., Dl or D2 medium which includes TGFp 1 or TGFp 3 , respectively, and 10 ng/ml bFGF
  • a Mab ADCB medium - based culture medium which includes a TGFp 1 or TGFp 3 isoform and bFGF at a concentration of 8 ng/ml was also capable of maintaining hESCs in the undifferentiated state for at least 5 passages (data not shown).
  • the bFGF which is included in TGFp-containing culture medium of this aspect of the present invention is provided at a concentration of at least 2 ng/ml, at least 3 ng, at least 4 ng/ml, at least 5 ng/ml, at least 6 ng/ml, at least 7 ng, at least 8 ng/ml, at least 9 ng/ml, at least 10 ng/ml.
  • the culture medium of this aspect of the present invention is devoid of a protein carrier (i.e., protein carrier-free).
  • a protein carrier refers to a protein which acts in the transfer of proteins or nutrients (e.g., minerals such as zinc) to the cells in the culture.
  • protein carriers can be, for example, albumin (e.g., bovine serum albumin), Albumax (lipid enriched albumin) or plasmanate (human plasma isolated proteins). Since these carriers are derived from either human or animal sources their use in hESCs cultures is limited by batch-specific variations and/or exposure to pathogens. On the other hand, the recombinant human albumin, which is substantially pure and devoid of animal contaminants is highly expensive, thus not commonly used in hESCs cultures. Thus, a culture medium which is devoid of a protein carrier is highly advantageous since it enables a truly defined medium that can be manufacture from recombinant or synthetic materials.
  • the culture medium of this aspect of the present invention is also devoid of serum (i.e., serum-free) or serum replacement (i.e., serum replacement-free).
  • serum or serum replacement are added to most culture media which are designed for culturing stem cells, and particularly, embryonic stem cells, in order to provide the cells with the optimal environment, similar to that present in vivo (i.e., within the organism from which the cells are derived, e.g., a blastocyst of an embryo or an adult tissue of a postnatal individual).
  • serum which is derived from either an animal source (e.g., bovine serum) or a human source (human serum) is limited by the significant variations in serum components between individuals and the risk of having xeno contaminants (in case of an animal serum is used)
  • the use of the more defined composition such as the currently available serum replacementTM (Gibco-Invitrogen Corporation, Grand Island, NY USA) may be limited by the presence of Albumax (Bovine serum albumin enriched with lipids) which is from an animal source within the composition (International Patent Publication No. WO 98/30679 to Price, P.J. et al).
  • a culture medium which comprises a TGF ⁇ isoform as described hereinabove and is devoid of serum, serum replacement and a protein carrier is highly desirable for both cell-based therapy and pharmaceutical industry, e.g., for pharmaceutical screening, identification of drug targets and cell-based compound delivery.
  • the culture medium of this aspect of the present invention is capable of expanding the stem cells while maintaining them in the undifferentiated state.
  • expanding refers to obtaining a plurality of cells from a single or a population of stem cells.
  • expanding embryonic stem cells refers also to increasing the number of embryonic stem cells over the culturing period.
  • the number of stem cells which can be obtained from a single stem cell depends on the proliferation capacity of the stem cell.
  • the proliferation capacity of a stem cell can be calculated by the doubling time of the cell (i.e., the time needed for a cell to undergo a mitotic division in the culture) and the period the stem cell culture can be maintained in the undifferentiated state (which is equivalent to the number of passages multiplied by the days between each passage).
  • hESCs could be maintained in the undifferentiated state in the presence of the Dl TGF ⁇ -containing culture medium for at least 53 passages. Given that each passage occurs every 4-7 days, the hESCs were maintained for 265 days (i.e., 6360 hours). Given that the hESCs doubling time was 36 hours, a single hESC cultured under these conditions could be expanded to give rise to 2 176 (i.e., 9.57 x 10 52 ) hESCs.
  • the stem cells which are maintained and expanded in the culture medium of the present invention exhibit stable karyotype (chromosomal stability) while in culture.
  • hESCs cultured in the presence of an IL6RIL6- containing medium e.g., CMlOO
  • a TGF ⁇ -containing medium e.g., Dl, D2 or HAl 6
  • a culture medium which includes the IL6RIL6 chimera is also capable of maintaining human ESCs in the undifferentiated state.
  • Humphrey R., et al., (2004) which failed to maintain hESCs in the undifferentiated state when using the "hyper IL6" complex and thus concluded that maintenance of pluropotency in human ESCs is STAT independent.
  • Humphrey R., et al., (2004) teaches away the present invention.
  • a culture system based on a f ⁇ bronectin or laminin feeder layer-free matrix and a culture medium which includes the IL6R ⁇ L6 chimera, serum replacement and bFGF was capable of maintaining hESCs in the undifferentiated state for at least 43 (on a fibronectin matrix) or 7 (on a laminin matrix) passages while preserving all hESCs characteristics and pluripotency.
  • Example 1 of the Examples section which follows, a medium containing serum replacement, bFGF and the unconjugated chimera components, i.e., IL-6 (GenBank Accession No. CAG29292) and soluble IL-6 receptor (GenBank Accession No. AAH89410), failed to support hESC prolonged culture and resulted in differentiation of hESCs within 3-5 passages.
  • hESCs cultured in a medium containing the IL6RIL6 chimera and serum replacement, in the absence of bFGF exhibited low proliferation capacity and could not be maintained in culture beyond 1-2 passages.
  • these results demonstrate, for the first time, that hESCs can be cultured and maintained in the undifferentiated state in a feeder-layer free culture system in the presence of a culture medium which comprises the IL6RIL6 chimera.
  • a culture medium which comprises an IL6RIL6 chimera, wherein the culture medium is capable of maintaining human embryonic stem cells in an undifferentiated state.
  • IL6RIL6 refers to a chimeric polypeptide which comprises the soluble portion of interleukin-6 receptor (IL-6-R, e.g., the human IL-6- R as set forth by GenBank Accession No. AAH89410) (e.g., a portion of the soluble IL6 receptors as set forth by amino acids 112-355 of GenBank Accession No.
  • the IL6RIL6 chimera used by the method according to this aspect of the present invention is capable of supporting the undifferentiated growth of human embryonic stem cells, while maintaining their pluripotent capacity.
  • the two functional portions i.e., the IL6 and its receptor
  • the two functional portions can be directly fused (e.g., attached or translationally fused, i.e., encoded by a single open reading frame) to each other or conjugated (attached or translationally fused) via a suitable linker (e.g., a polypeptide linker).
  • a suitable linker e.g., a polypeptide linker
  • the IL6RIL6 chimeric polypeptide exhibits a similar amount and pattern of glycosylation as the naturally occurring IL6 and IL6 receptor.
  • a suitable IL6RIL6 chimera is as set forth in SEQ ID NO:31 and in Figure 11 of WO 99/02552 to Revel M., et al., which is fully incorporated herein by reference.
  • any of the proteinaceous factors used in the culture medium of the present invention can be recombinantly expressed or biochemically synthesized.
  • naturally occurring proteinaceous factors such as bFGF and TGF ⁇ can be purified from biological samples (e.g., from human serum, cell cultures) using methods well known in the art.
  • Biochemical synthesis of the proteinaceous factors of the present invention can be performed using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis. Recombinant expression of the proteinaceous factors of the present invention
  • IL6RIL6 chimera can be generated using recombinant techniques such as described by Bitter et al, (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 3:1671- . 1680, Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI. Cell. Biol.
  • IL6RIL6 chimera can be generated as described in PCT publication WO 99/02552 to Revel M., et al. and Chebath J,, et al., 1997, which are fully incorporated herein by reference.
  • a polynucleotide sequence encoding the IL6RIL6 chimera (e.g., the polypeptide set forth by SEQ ID NO:31) is preferably ligated into a nucleic acid construct suitable for expression in a host cell [i.e., a cell in which the polynucleotide encoding the polypeptide-of-choice (e.g., the IL6RIL6 chimera) is expressed].
  • the host cell employed is a eukaryotic host cell, more preferably a mammalian host cell such as human cell or CHO cell).
  • mammalian expression vectors For expression in mammalian cells [e.g., CHO cells, human HEK 293 cells (ATCC CRL 1573)] a number of mammalian expression vectors can be used. Examples include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), ⁇ GL3, ⁇ ZeoSV2(+/-), ⁇ SecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRe ⁇ 5, DH26S, DHBB, pNMTl, pNMT41, pNMTSl, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • mammalian expression vectors include, but are not limited to,
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pB V-I MTHA 5 and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , ⁇ MTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Transformed cells are cultured under effective conditions, which allow for the expression of high amounts of the recombinant polypeptide (e.g., the IL6RIL6 chimera). Following a predetermined time in culture, recovery of the recombinant polypeptide is effected.
  • the phrase "recovery of the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
  • polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
  • the polypeptide of the present invention e.g., the IL6RIL6 chimera
  • the phrase "substantially pure” refers to a purity that allows for the effective use of the polypeptide of the present invention (e.g., the IL6RIL6 chimera) in maintaining the human embryonic stem cells in an undifferentiated state while in culture.
  • a medium supplemented with the 100 or 300 ng/ml of the IL6RIL6 chimera enabled the prolonged culturing of hESCs in the undifferentiated state for at least 43 passages, while preserving normal karyotype and pluripotent capacity [as evidenced by the formation of embryoid bodies (EBs) which express markers of all three embryonic germ cells].
  • EBs embryoid bodies
  • the IL6RIL6 chimera which is included in the culture medium of this aspect of the present invention is provided at a concentration of at least 25 ng/ml, at least 50 ng/ml, at least 100 ng/ml, preferably, at least 200 ng/ml, preferably, at least 300 ng/ml.
  • concentration of the IL6RIL6 chimera can vary depending on the purity of the chimeric polypeptide following its synthesis or recombinant expression and those of skills in the art -are capable of adjusting the optimal concentration depending on such purity.
  • the IL6RIL6-containing culture medium of this aspect of the present invention includes at least 2 ng/ml bFGF, at least 3 ng/ml, at least 4 ng/ml, at least 5 ng/ml, at least 6 ng/ml, at least 7 ng, at least 8 ng/ml, at least 9 ng/ml, at least 10 ng/ml bFGF.
  • a culture medium which includes 4 ng/ml bFGF along with 100 ng/ml of the IL6RIL6 chimera was capable of maintaining hESCs in an undifferentiated state for at least 43 passages.
  • a culture medium can also include serum (e.g., such as human serum) instead of serum replacement and yet maintain hESCs in culture in the undifferentiated state.
  • serum or serum replacement which are included in the IL ⁇ RIL-containing culture medium of this aspect of the present invention can be provided at various concentrations, such as a concentration of at least 10 %, e.g., a concentration of at least 15 %, at least 20 %, at least 25 % or at least 30 %.
  • the IL6RIL6-containing culture medium of this aspect of the present invention is preferably devoid of serum or serum replacement.
  • a non-limiting example of such a culture medium can be the HACMlOO culture medium described in Examples 2 and 4 of the Examples section which follows.
  • any of the culture media described hereinabove, which are based on the TGF ⁇ isoform or the IL6RIL6 chimera present, for the first time, a well-defined, xeno-free culture medium which is highly suitable for culturing hESCs for applications such as cell-based therapy and for use in the pharmaceutical industry.
  • a culture medium which is devoid of serum ⁇ i.e., serum-free), xeno contaminant ⁇ i.e., xeno-free), feeder layers ⁇ i.e., feeder layer-free) and protein carrier ⁇ i.e., protein carrier-free) and yet capable of maintaining stem cells in an undifferentiated state.
  • stem cells such as human embryonic stem cells can be expanded using any of the culturing media described hereinabove and be maintained in the undifferentiated state while in culture.
  • a cell culture which comprises the stem cell and any of the culture media described hereinabove, while the culture medium is capable of maintaining said stem cells in an undifferentiated state.
  • a method of expanding and maintaining stem cells in an undifferentiated state is effected by culturing the stem cells in any of the culture media described hereinabove, thereby expanding and maintaining the stem cells in the undifferentiated state.
  • Culturing according to this aspect of the present invention is effected by plating the stem cells onto a matrix in a cell density which promotes cell survival and proliferation but limits differentiation. Typically, a plating density of between about 15,000 cells/cm 2 and about 3,000,000 cells/cm 2 is used. It will be appreciated that although single-cell suspensions of stem cells are usually seeded, small clusters may also be used. To this end, enzymatic digestion (such as with type IV collagenase) utilized for cluster disruption (see Examples 1 and 2 of the Examples section which follows) is terminated before stem cells become completely dispersed and the cells are triturated with a pipette such that clumps (i.e., 10-200 cells) are formed.
  • a pipette such that clumps (i.e., 10-200 cells) are formed.
  • stem cells can be cultured on feeder cells or on feeder- layer free culturing systems using a matrix instead of a feeder cell layer.
  • matrix refers to any substance to which the stem cells can adhere and which therefore can substitute the cell attachment function of feeder cells.
  • Such a matrix typically contains extracellular components to which the stem cells can attach and thus it provides a suitable culture substrate.
  • extracellular matrix components derived from basement membrane or extracellular matrix components that form part of adhesion molecule receptor-ligand couplings.
  • suitable matrices which can be used by the method of this aspect of the present invention include Matrigel ® (Becton Dickinson, USA), laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
  • the matrix is preferably derived from a human source or synthesized using recombinant techniques.
  • Such matrices include, for example, human-derived fibronectin, recombinant fibronectin, human-derived laminin, foreskin fibroblast matrix or a synthetic fibronectin matrix which can be obtained from Sigma, St. Louis, MO, USA or can be produced using known recombinant DNA technology.
  • Preferred matrices of the present invention are fibronectin derived matrices.
  • stem cells cultured using the teachings of the present invention can be expanded while maintaining in the undifferentiated state.
  • culturing of hESCs on a MatrigelTM in the presence of the TGF ⁇ -containing culture medium (the Dl medium) for at least 53 passages (8-9 months) resulted in an expansion factor of 2 180 (i.e., 1.5 x 10 54 ) given that the hESC doubling time is 36 hours and that passaging occurs every 4-6 days.
  • ESCs can be expanded in a suspension culture devoid of substrate adherence and as such can be maintained in the undifferentiated, pluripotent state.
  • suspension culture refers to a culture in which the embryonic stem cells are suspended in a medium rather than adhering to a surface.
  • the culture of the present invention is "devoid of substrate adherence" in which the stem cells (e.g., ESCs) are capable of expanding without adherence to an external substrate such as components of extracellular matrix, a glass microcarrier or beads.
  • the stem cells e.g., ESCs
  • an external substrate such as components of extracellular matrix, a glass microcarrier or beads.
  • hESCs cultured in a suspension culture devoid of substrate adherence can be expanded for at least 17 passages in the CMlOOF medium and maintain their undifferentiated, pluripotent state (Example 4 and data not shown).
  • stem cell growth is monitored to determine their differentiation state.
  • the differentiation state can be determined using various approaches including, for example, morphological evaluation (e.g., as shown in Figures la-f, 5a-d and 9a-g) and/or detection of the expression pattern of typical markers of the undifferentiated state using immunological techniques such as flow cytometry for membrane-bound markers, immunohistochemistry or immunofluorescence for extracellular and intracellular markers and enzymatic immunoassay, for secreted molecular markers.
  • morphological evaluation e.g., as shown in Figures la-f, 5a-d and 9a-g
  • detection of the expression pattern of typical markers of the undifferentiated state using immunological techniques such as flow cytometry for membrane-bound markers, immunohistochemistry or immunofluorescence for extracellular and intracellular markers and enzymatic immunoassay, for secreted molecular markers.
  • immunofluorescence employed on hESCs cultured according to the method of this aspect of the present invention revealed the expression of Oct4, stage-specific embryonic antigen (SSEA) 4, the tumour-rejecting antigen (TRA)-l-60 and TRA-I- 81 ( Figures 3a-c, 6a-c and 8a-c).
  • SSEA stage-specific embryonic antigen
  • TRA tumour-rejecting antigen
  • the level of transcripts of specific undifferentiation markers e.g., Oct 4, Nanog, Sox2, Rexl, Cx43, FGF4
  • differentiation markers e.g., albumin, glucagons, ⁇ -cardiac actin, ⁇ -globulin, Flkl, AC 133 and neurofilament
  • RNA-based techniques such as RT- PCR analysis (as shown in Figures 4a-l and 10a-d) and/or cDNA microarray analysis. Determination of ES cell differentiation can also be effected via measurements of alkaline phosphatase activity.
  • Undifferentiated human ES cells have alkaline phosphatase activity which can be detected by fixing the cells with 4 % paraformaldehyde and developing with the Vector Red substrate kit according to manufacturer's instructions (Vector Laboratories, Burlingame, California, USA).
  • a method of deriving an embryonic stem cell line is effected by: (a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing the embryonic stem cell in any of the culture media described hereinabove, thereby deriving the embryonic stem cell line.
  • embryonic stem cell line refers to embryonic stem cells which are derived from a single or a group of embryonic stem cells of a single organism (e.g., a single human blastocyst), and which are characterized by the ability to proliferate in culture while maintaining the undifferentiated state and the pluripotent capacity.
  • an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus can be performed using methods known in the art, as described hereinabove and in the "General Materials and Experimental Methods" of the Examples section which follows.
  • the zona pellucida is removed from a 5-7 day-old blastocyst using Tyrode's acidic solution (Sigma, St Louis MO, USA), the trophoblast layer is specifically removed either by immunosurgery or mechanically using 27g needles and the exposed ICM is either directly cultured in a suitable culture system (e.g., feeder layers, feeder-free matrix or a suspension culture) in the presence of any of the culture media described hereinabove (e.g., the CMlOOF, HA 16 or D2 medium) for 4-10 days (in case a preimplantation blastocyst is used) or subject to in vitro implantation by culturing the ICM for 6-8 days (to obtain cells of a 13 day-old blastocyst in case a post-implantation/pre-gastrulation blastocyst is used) on feeder layers or a feeder-free culturing system which allow implantation of the blastocyst to the surface, following which the implanted cells are
  • the genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation.
  • the single cell EG cells are then cultured in any of the culture media described hereinabove for 4-10 days).
  • the ESCs are further cultured in any of the culture media described hereinabove which allow expansion of the embryonic stem cells in the undifferentiated state, essentially as described hereinabove.
  • the cell culture of the present invention is characterized by at least 40 %, at least 50 %, at least 60 %, more preferably at least 70 %, more preferably at least 80 %, most preferably at least 85 % of undifferentiated stem cells.
  • an established embryonic stem cell line can be subject to freeze/thaw cycles without hampering the proliferative capacity of the cells in the undifferentiated state while preserving their pluripotent capacity.
  • hESCs were successfully frozen and thawed.
  • Figures 4a-l is described in Examples 1, 2 and 4 of the
  • hESCs which were expanded and maintained in any of the culture media described hereinabove are pluripotent ⁇ i.e., capable of differentiating into all cell types of the three embryonic germ layers, the ectoderm, the endoderm and the mesoderm) as evidenced in vitro (by the formation of EBs) and in vzvo (by the formation of teratomas).
  • hESCs cultured according to the teachings of the present invention can be used as a source for generating differentiated, lineage-specific cells.
  • Such cells can be obtained directly from the ESCs by subjecting the ESCs to various differentiation signals (e.g., cytokines, hormones, growth factors) or indirectly, via the formation of embryoid bodies and the subsequent differentiation of cells of the EBs to lineage-specific cells.
  • various differentiation signals e.g., cytokines, hormones, growth factors
  • a method of generating embryoid bodies from embryonic stem cells is effected by (a) culturing the embryonic stem cells in any of the culture media described hereinabove which is capable of maintaining the embryonic stem cells in an undifferentiated state, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting the expanded, undifferentiated embryonic stem cells to culturing conditions suitable for differentiating the embryonic stem cells to embryoid bodies; thereby generating the embryoid bodies from the embryonic stem cells.
  • embryonic bodies refers to morphological structures comprised of a population of ESCs, extended blastocyst cells (EBCs) and/or embryonic germ cells (EGCs) which have undergone differentiation.
  • EBCs extended blastocyst cells
  • EECs embryonic germ cells
  • EBs formation initiates folio whig the removal of differentiation blocking factors from ES cell cultures.
  • ESCs proliferate into small masses of cells which then proceed with differentiation.
  • a layer of endodermal cells is formed on the outer layer of the small mass, resulting in "simple EBs".
  • complex EBs are formed. Complex EBs are characterized by extensive differentiation of ectodermal and mesodermal cells and derivative tissues.
  • the method of this aspect of the present invention involves the culturing of ESCs in any of the culture media described hereinabove in order to obtain expanded, undifferentiated embryonic stem cells and then subjecting the expanded, undifferentiated ESCs to culturing conditions suitable for differentiating the ESCs to embryoid bodies.
  • Such culturing conditions are substantially devoid of differentiation inhibitory factors which were employed during step (a), e.g., a TGF ⁇ isoform or the IL6RIL6 chimera.
  • a culture medium suitable for EBs formation may include a basic culture medium (e.g., Ko-DMEM or DMEM/F12) supplemented with 20 % FBSd (HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, and 1 % non-essential amino acid stock.
  • a basic culture medium e.g., Ko-DMEM or DMEM/F12
  • Monitoring the formation of EBs can be effected by morphological evaluations (e.g., histological staining), determination of expression of differentiation- specific markers [e.g., using immunological techniques or RNA-based analysis (e.g.,
  • cells harvested from EBs according to the method of this aspect of the present invention exhibited markers of all three embryonic germ layers, such as albumin and glucagon (typical of the embryonic endoderm), ⁇ -cardiac actin, ⁇ -globulin and FIk 1 (typical of the embryonic mesoderm), and AC 133 and neurofilament (NFH) (typical of the embryonic ectoderm).
  • markers of all three embryonic germ layers such as albumin and glucagon (typical of the embryonic endoderm), ⁇ -cardiac actin, ⁇ -globulin and FIk 1 (typical of the embryonic mesoderm), and AC 133 and neurofilament (NFH) (typical of the embryonic ectoderm).
  • cells of the EBs can be further subjected to culturing conditions suitable for lineage-specific cells.
  • the method of this aspect of the present invention further includes step (c) of subjecting cells of the embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage- specific cells from the embryonic stem cells.
  • culturing conditions suitable for differentiating and/or expanding lineage specific cells refers to a combination of culture system, e.g., feeder cell layers, feeder-free matrix or a suspension culture and a culture medium which are suitable for the differentiation and/or expansion of specific cell lineages derived from cells of the EBs.
  • culture system e.g., feeder cell layers, feeder-free matrix or a suspension culture and a culture medium which are suitable for the differentiation and/or expansion of specific cell lineages derived from cells of the EBs.
  • Non-limiting examples of such culturing conditions are further described hereinunder.
  • the method of this aspect of the present invention further includes isolating lineage specific cells following step (b).
  • the phrase "isolating lineage specific cells” refers to the enrichment of a mixed population of cells in a culture with cells predominantly displaying at least one characteristic associated with a specific lineage phenotype. It will be appreciated that all cell lineages are derived from the three embryonic germ layers. Thus, for example, hepatocytes and pancreatic cells are derived from the embryonic endoderm, osseous, cartilaginous, elastic, fibrous connective tissues, myocytes, myocardial cells, bone marrow cells, vascular cells (namely endothelial and smooth muscle cells), and hematopoietic cells are differentiated from embryonic mesoderm and neural, retina and epidermal cells are derived from the embryonic ectoderm.
  • isolating is effected by sorting of cells of the EBs via fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • EBs are disaggregated using a solution of Trypsin and EDTA (0.025 % and 0.01 %, respectively), washed with 5 % fetal bovine serum (FBS) in phosphate buffered saline (PBS) and incubated for 30 min on ice with fluorescently-labeled antibodies directed against cell surface antigens characteristics to a specific cell lineage.
  • FBS fetal bovine serum
  • PBS phosphate buffered saline
  • endothelial cells are isolated by attaching an antibody directed against the platelet endothelial cell adhesion molecule- 1 (PECAMl) such as the fluorescently-labeled PECAMl antibodies (30884X) available from PharMingen (PharMingen, Becton Dickinson Bio Sciences, San Jose, CA, USA) as described in Levenberg, S. et al., (Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396).
  • PECAMl platelet endothelial cell adhesion molecule- 1
  • Hematopoietic cells are isolated using fluorescently-labeled antibodies such as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CDl 17-PE, CD15-FITC, class I-FITC, all of which IgGl are available from PharMingen, CD133/1-PE (IgGl) (available from Miltenyi Biotec, Auburn, CA), and glycophorin A-PE (IgGl), available from Immunotech (Miami, FL). Live cells (i.e., without fixation) are analyzed on a FACScan (Becton Dickinson Bio Sciences) by using propidium iodide to exclude dead cells with either the PC-LYSIS or the CELLQUEST software.
  • fluorescently-labeled antibodies such as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CDl 17-PE, CD15-FITC, class I-FITC, all of which IgG
  • isolated cells can be further enriched using magnetically-labeled second antibodies and magnetic separation columns (MACS, Miltenyi) as described by Kaufman, D.S. et al., (Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 2001, 98: 10716-10721).
  • MCS magnetically-labeled second antibodies and magnetic separation columns
  • isolating is effected by a mechanical separation of cells, tissues and/or tissue-like structures contained within the EBs.
  • beating cardiomyocytes can be isolated from EBs as disclosed in U.S. Pat. Appl. No. 20030022367 to Xu et al.
  • Four-day-old EBs of the present invention are transferred to gelatin-coated plates or chamber slides and are allowed to attach and differentiate.
  • Spontaneously contracting cells which are observed from day 8 of differentiation, are mechanically separated and collected into a 15-mL tube containing low-calcium medium or PBS.
  • Cells are dissociated using Collagenase B digestion for 60-120 minutes at 37 °C, depending on the Collagenase activity. Dissociated cells are then resuspended in a differentiation KB medium (85 mM KCI, 30 mM K 2 HPO 4 , 5 mM MgSO 4 , 1 mM EGTA, 5 mM creatine, 20 mM glucose, 2 mM Na 2 ATP, 5 mM pyruvate, and 20 mM taurine, buffered to pH 7.2, Maltsev et al., Circ. Res. 75:233, 1994) and incubated at 37 °C for 15-30 min. Following dissociation cells are seeded into chamber slides and cultured in the differentiation medium to generate single cardiomyocytes capable of beating.
  • a differentiation KB medium 85 mM KCI, 30 mM K 2 HPO 4 , 5 mM MgSO 4 , 1 mM EGTA, 5 mM creat
  • isolating is effected by subjecting the EBs to differentiation factors to thereby induce differentiation of the EBs into lineage specific differentiated cells.
  • EBs of the present invention are cultured for 5-12 days in tissue culture dishes including DMEM/F-12 medium with 5 mg/ml insulin, 50 mg/ml transferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFn medium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102).
  • tissue culture dishes including DMEM/F-12 medium with 5 mg/ml insulin, 50 mg/ml transferrin, 30 nM selenium chloride, and 5 mg/ml fibronectin (ITSFn medium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102).
  • the resultant neural precursors can be further transplanted to generate neural cells in vivo (Br ⁇ stle, O. et al., 1997. In w ⁇ r ⁇ -generated neural precursors participate in mammalian brain development. Proc. Natl. Acad. Sci. USA. 94: 14809-14814). It will be appreciated that prior to their transplantation, the neural precursors are trypsinized and triturated to single-cell suspensions in the presence of 0.1 % DNase.
  • EBs of the present invention can differentiate to oligodendrocytes and myelinate cells by culruring the cells in modified SATO medium, i.e., DMEM with bovine serum albumin (BSA), pyruvate, progesterone, putrescine, thyroxine, triiodothryonine, insulin, transferrin, sodium selenite, amino acids, neurotrophin 3, ciliary neurotrophic factor and Hepes (Bottenstein, J. E. & Sato, G. H., 1979, Proc. Natl. Acad. Sci. USA 76, 514-517; Raff, M. C, Miller, R. H., & Noble, M., 1983, Nature 303: 390-396].
  • modified SATO medium i.e., DMEM with bovine serum albumin (BSA), pyruvate, progesterone, putrescine, thyroxine, triiodothryonine, insulin, transferrin,
  • EBs are dissociated using 0.25 % Trypsin/EDTA (5 min at 37 °C) and triturated to single cell suspensions.
  • Suspended cells are plated in flasks containing SATO medium supplemented with 5 % equine serum and 5 % fetal calf serum (FCS).
  • FCS fetal calf serum
  • the flasks are gently shaken to suspend loosely adhering cells (primarily oligodendrocytes), while astrocytes are remained adhering to the flasks and further producing conditioned medium.
  • Primary oligodendrocytes are transferred to new flasks containing SATO medium for additional two days.
  • oligospheres are either partially dissociated and resuspended in SATO medium for cell transplantation, or completely dissociated and a plated in an oligosphere-conditioned medium which is derived from the previous shaking step [Liu, S. et al., (2000). Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl. Acad. Sci. USA. 97: 6126-6131].
  • two-week-old EBs of the present invention are transferred to tissue culture dishes including DMEM medium supplemented with 10 % FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 20 % (v/v) WEHI-3 cell-conditioned medium and 50 ng/ml recombinant rat stem cell factor (rrSCF, Tsai, M. et al., 2000.
  • rrSCF recombinant rat stem cell factor
  • hemato-lymphoid cells from the EBs of the present invention, 2-3 days-old EBs are transferred to gas-permeable culture dishes in the presence of 7.5 % CO 2 and 5 % O 2 using an incubator with adjustable oxygen content. Following 15 days of differentiation, cells are harvested and dissociated by gentle digestion with Collagenase (0.1 unit/mg) and Dispase (0.8 unit/mg), both are available from F.Hoffman-La Roche Ltd, Basel, Switzerland. CD45-positive cells are isolated using anti-CD45 monoclonal antibody (mAb) M1/9.3.4.HL.2 and paramagnetic microbeads (Miltenyi) conjugated to goat anti-rat immunoglobulin as described in Potocnik, AJ.
  • mAb monoclonal antibody
  • Miltenyi paramagnetic microbeads
  • the isolated CD45- ⁇ ositive cells can be further enriched using a single passage over a MACS column (Miltenyi).
  • the culturing conditions suitable for the differentiation and expansion of the isolated lineage specific cells include various tissue culture media, growth factors, antibiotic, amino acids and the like and it is within the capability of one skilled in the art to determine which conditions should be applied in order to expand and differentiate particular cell types and/or cell lineages.
  • lineage specific cells can be obtained by directly inducing the expanded, undifferentiated ESCs to culturing conditions suitable for the differentiation of specific cell lineage.
  • EBs of the present invention can be used to generate lineage-specific cell lines which are capable of unlimited expansion in culture.
  • Cell lines of the present invention can be produced by immortalizing the EB- derived cells by methods known in the art, including, for example, expressing a telomerase gene in the cells (Wei, W. et al., 2003. MoI Cell Biol. 23: 2859-2870) or co-culturing the cells with NIH 3T3 hph-HOXl 1 retroviral producer cells (Hawley, R.G. et al., 1994. Oncogene 9: 1-12). It will be appreciated that since the lineage-specific cells or cell lines obtained according to the teachings of the present invention are developed by differentiation processes similar to those naturally occurring in the human embryo they can be further used for human cell-based therapy and tissue regeneration.
  • the present invention envisages the use of the expanded and/or differentiated lineage-specific cells or cell lines of the present invention for treating a disorder requiring cell replacement therapy.
  • oligodendrocyte precursors can be used to treat myelin disorders (Repair of myelin disease: Strategies and progress in animal models. Molecular Medicine Today. 1997. pp. 554-561), chondrocytes or mesenchymal cells can be used in treatment of bone and cartilage defects (U.S. Pat. No. 4,642,120) and cells of the epithelial lineage can be used in skin regeneration of a wound or burn (U.S. Pat. No. 5,716,411).
  • genetic disorders in which a specific gene product is missing e.g., lack of the CFTR gene-product in cystic fibrosis patients (Davies JC 5 2002. New therapeutic approaches for cystic fibrosis lung disease.
  • ESC-derived cells are preferably manipulated to over- express the mutated gene prior to their administration to the individual. It will be appreciated that for other disorders, the ESC-derived cells should be manipulated to exclude certain genes.
  • Over-expression or exclusion of genes can be effected using knock-in and/or knock-out constructs [see for example, Fukushige, S. and Ikeda, J. E.: Trapping of mammalian promoters by Cre-lox site-specific recombination. DNA Res 3 (1996) 73-
  • the lineage specific cells of the present invention can also be utilized to prepare a cDNA library.
  • mRNA is prepared by standard techniques from the lineage specific cells and is further reverse transcribed to form cDNA.
  • the cDNA preparation can be subtracted with nucleotides from embryonic fibroblasts and other cells of undesired specificity, to produce a subtracted cDNA library by techniques known in the art.
  • the lineage specific cells of the present invention can be used to screen for factors (such as small molecule drugs, peptides, polynucleotides, and the like) or conditions (such as culture conditions or manipulation) that affect the differentiation of lineage precursor to terminally differentiated cells.
  • factors such as small molecule drugs, peptides, polynucleotides, and the like
  • conditions such as culture conditions or manipulation
  • growth affecting substances, toxins or potential differentiation factors can be tested by their addition to the culture medium.
  • MEFs mouse embryonic fibroblasts
  • hESC basic culture medium consisting of 85 % Ko-DMEM, supplemented with 15 % serum replacement (SR), 2 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1 % non-essential amino acid stock, and 4 ng/ml bFGF (all Gibco Invitrogen Corporation products, Grand Island NY, USA).
  • hESCs were transferred to 50 ⁇ g per 10 cm 2 fibronectin-covered plates (human plasma fibronectin, Chemicon International, Temecula CA, USA) in the presence of the "hESC basic culture medium" with the following combinations of cytokines:
  • CM6 medium - 0.3 ng/ml Interleukin-6 (IL6) and 0.5 ng/ml IL6 soluble receptor (both from R&D Systems Minneapolis MN, USA);
  • IL-6-IL-6 receptor (IL6RIL6) chimera - 50 ng/ml, 100 ng/ml, 200 ng/ml or 300 ng/ml of IL6RIL6 chimera (Chebath J, et al., 1997 and WO 99/02552 to Revel M., et al. SEQ ID NO:31).
  • this medium is also called CMlOO.
  • Control medium - 4 ng/ml bFGF (Gibco Invitrogen Corporation,
  • Karyotype analysis Karyotype analysis (G-banding) was performed on at least 20 cells from each sample, two samples per test, as previously described [Amit et al, 2003]. Karyotypes were analyzed and reported according to the "International System for Human Cytogenetic Nomenclature" (ISCN).
  • ISCN International System for Human Cytogenetic Nomenclature
  • EB formation For the formation of EBs, one to four confluent wells were used in a six-well plate (40 cm 2 ). ESCs were removed from their culture dish using 1 mg/ml type IV collagenase, further broken into small clumps using 1000 ⁇ l Gilson pipette tips, and cultured in suspension in 58 mm petri dishes (Greiner, Frickenhausen, Germany).
  • EBs were grown in medium consisting of 80 % Ko- DMEM 5 supplemented with 20 % FBSd (HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, and 1 % non-essential amino acid stock (all but FBSd from Gibco Invitrogen Corporation, Grand Island NY, USA). 10 day-old EBs were harvested for RNA isolation and histological examination.
  • PCR reactions included denaturation for 5 minutes at 94 °C followed by repeated cycles of 94 °C for 30 seconds, annealing for 30 seconds at a temperature specified in Table 1, hereinbelow, and extension at 72 °C for 30 seconds.
  • PCR primers and reaction conditions used are described in Table 1, hereinbelow.
  • PCR products were size-fractionated using 2 % agarose gel electrophoresis. DNA markers were used to confirm the size of the resultant fragments.
  • IL6RIL6 chimera Medium supplemented with a combination of 100 ng/ml IL6RIL6 chimera and 4 ng/ml bFGF is most suitable to support undifferentiated proliferation of ItESCs on a feeder-free culture system -
  • concentrations of the IL6RIL6 chimera were tested for the ability to support the feeder-layer free culture of hESCs.
  • two measures were used to estimate the ability of the hESCs to grow in the feeder-free culture system, namely percentage of differentiation and rate of growth.
  • the medium supplemented with the combination of 100 ng/ml IL6RIL6 chimera and 4 ng/ml bFGF was found to be the most suitable to support undifferentiated feeder- layer free hESC proliferation.
  • the hESCs could be cultured continuously in feeder-layer free conditions for at least 43 passages.
  • hESCs cultured in these conditions maintained their ESC features, including undifferentiated proliferation, karyotype stability and pluripotency as was tested following 28 passages. Based on morphology, the background spontaneous differentiation rates were about 15 %, similar to those occurring in other feeder layer-free culture methods [Xu et al, 2001, 2005; Amit et al, 2004].
  • Feeder-free culture system in the presence of a culture medium containing the IL6RIL6 chimera supports hESCs with typical morphology -
  • a culture medium containing the IL6RIL6 chimera supports hESCs with typical morphology -
  • no morphological differences could be observed between colonies grown in the feeder layer-free culture system (a fibronectin matrix and a medium supplemented with IL6RIL6 chimera and bFGF) and those grown on MEFs ( Figures la-f and data not shown).
  • the morphological features at the single-cell level remained unchanged, the cells remained small and round, and exhibited high nucleus-to-cytoplasm ratio, with a notable presence of one to three nucleoli and with typical spacing between the cells ( Figures la-f).
  • the hESC population doubling time was similar to that observed when grown on MEFs (about 36 hours), and in the present feeder-free conditions the cells could be passaged routinely every four to six days, at a ratio of 1/2 or 1/3, similar to the splitting ratio employed when the hESCs are cultured on MEFs.
  • the cells were passaged at the same seeding efficiency as with MEF of about 1 million cells per 10 cm 2 , with the same viability rate of over 90 %. Using 15 % SR and 10 % DMSO, cells were successfully frozen and thawed.
  • the fibronectin feeder-layer free culture system supplemented with a medium containing the IL6RIL6 chimera supports hESCs with normal karyotype or at least 23 passages - Karyotype analysis by Giemsa banding was carried out on two separate cultures, representing the two medium conditions: medium supplemented with 4 ng/ml bFGF and either 100 ng/ml or 300 ng/ml IL6RIL6 chimera 23 passages after transferring the cells into the feeder-layer environment which is based on fibronectin. At least 20 cells were tested from each sample, 40 cells from each medium combination. All examined cells were found to sustain normal karyotype of 46, XX and 46,XY for cell lines 13 and 16, respectively.
  • hESCs cultured on the fibronectin feeder-free culture system for 27 passages with medium supplemented with 100 ng/ml or 300 ng/ml of the IL6RIL6 chimera were found to be strongly positive to surface markers TRA-1-60 (Figure 3a) Oct4 ( Figure 3b), SSEA4 ( Figure 3c), and TRA-1-81 (data not shown). As in other primate ES cells, staining with S SE A3 was weak and negative for SSEAl (data not shown).
  • the IL6RIL6 chimera medium is capable of supporting pluripotent hESCs as evident by the in vitro differentiation into embryoid bodies (EBs) -
  • EBs embryoid bodies
  • the developmental potential of the cells after prolonged culture in feeder layer-free conditions was examined in vitro by the formation of EBs.
  • hESCs formed EBs similar to those created by ES cells grown on MEFs.
  • stem cells differentiated into cell types representative of the three embryonic germ layers.
  • EBs formed from the hESCs cultured on the IL6RIL6 chimera expressed typical differentiation markers of all embryonic cell lineages —
  • RT-PCR analysis was performed using primers specific to both differentiation and undifferentiated cells (primers employed are listed in Table 1, hereinabove).
  • Human ESCs like mouse ES cells, are traditionally cultured with MEFs, which may expose them to animal pathogens.
  • MEFs mouse ES cells
  • the present inventors have demonstrated a defined medium and feeder layer-free culture system based on the use of serum replacementTM, bFGF, IL6RIL6 chimera and human fibronectin as a matrix substitute.
  • IL6RIL6 chimera concentrations of the IL6RIL6 chimera were tested for their ability to maintain hESCs in an undifferentiated state.
  • the most suitable medium was that supplemented with 100 ng/ml of the IL6RIL6 chimera together with 4 ng/ml bFGF, in which the three transferred hESC lines continued to proliferate while retaining normal hESC features throughout the prolonged culture.
  • ES cells cultured in these conditions maintained all characteristics of ES cells. After prolonged culture of up to 28 passages, the cells remained undifferentiated, as demonstrated by the colony and single cell morphology, and by the expression of surface markers typical of undifferentiated primate ESCs [Thomson et al, 1995, 1996, 1998; Reubinoff et al, 2000].
  • hESCs expressed specific markers for the undifferentiated stage such as Oct 4, Sox 2, Rexl and FGF4, as demonstrated by immunofluorescence (for Oct4) and RT-PCR analyses (for Oct4, Sox 2, Rexl and FGF4).
  • the IL6RIL6 chimera was demonstrated as the most potent factor in supporting the feeder layer-free isolation of mouse ESC lines (Nichols et al, 1994). Previous studies did not demonstrate a significant effect of the IL-6 family, including a fusion protein of portions of IL-6 and the IL-6 receptor, on the self-maintenance of undifferentiated liESCs [Daheron et al, 2004; Humphrey et al, 2004; Sato et al, 2004]. The specific conditions for hESC cultures described here may explain why the IL6RIL6 chimera was effective in supporting the proliferation of hESC with minimal differentiation.
  • fibronectin substrate as a matrix (and not mouse laminin) and another being the precise schedule of hESC passaging. Further research is required to elucidate the underlying mechanisms of action of the IL6RIL6 chimera at the level of signal transduction, its time course and intensity at which different pathways (JAK/STAT, PI3 kinase, MAPK, see Hirano et al, 1997) are activated.
  • hESCs Future clinical uses of hESCs will require a reproducible, well-defined and xeno-free culture system.
  • serum replacement used in the present study is considered such, it contains "Albumax" which is a lipid-enriched bovine serum albumin and, therefore, is not animal-free.
  • the well-defined conditioned media demonstrated in the present study are suitable for culturing hESCs and may be advantageous for undertaking research on the mechanisms of ESC self-maintenance, especially of the possible roles of the LIF/STAT3 pathway and various integrins as fibronectin receptors.
  • Other studies using hESCs, such as the research on differentiation pathways and mechanisms, will benefit from the availability of a well- defined and reproducible culture system.
  • This culture system is a further step forward towards fully defined culture conditions for hESCs, and promotes the development of a xeno-free culture system for hESCs.
  • MEFs mouse embryonic fibroblasts
  • basic hESC culture medium consisting of 85 % DMEM/F12 (Biological Industries, Biet Haemek, Israel) supplemented with 15 % serum replacement (SR), 2 mM L-glutamine, 0.1 mM ⁇ -mercaptoethanol, 1 % non- essential amino acid stock, and 4 ng/ml basic fibroblast growth factor (bFGF) (all Gibco Invitrogen Corporation products, Grand Island NY, USA).
  • SR serum replacement
  • bFGF basic fibroblast growth factor
  • Fibronectin feeder-free culture system 50 ⁇ g per 10 cm 2 fibronectin- covered plates (human plasma fibronectin, Chemicon International, Temecula CA, USA);
  • Dl medium - Mab ADCB medium (HyClone, Utah, USA) supplemented with 2 mM L-glutamine (Invitrogen Corporation products, Grand Island NY, USA), 0.12 ng/ml TGFp 1 (from R&D Systems Minneapolis MN, USA), and 10 ng/ml bFGF (Invitrogen Corporation products, Grand Island NY, USA).
  • D2 medium - Mab ADCB medium (HyClone, Utah, USA) supplemented with 2 mM L-glutamine (Invitrogen Corporation products, Grand Island NY, USA), 2 ng/ml TGF ⁇ 3 and 10 ng/ml bFGF (Invitrogen Corporation products, Grand Island NY, USA).
  • HAU medium - 96 % DMEM/F12 (Biological Industries, Biet Haemek,
  • the ITS premix is a XlOOO stock solution obtained from BD Biosciences, Bedford, MA, USA and consists of 12.5 mg Insulin, 12.5 mg Transferrin and 12.5 mg Selenius acid], 2 mM L- glutamine, 2 ng/ml TGF ⁇ 3 (from R&D Systems Minneapolis MN, USA), 4 ng/ml bFGF, 500 ng/ml ascorbic acid (Sigma, Steinheim, Germany), and a 1:1000 dilution of a lipid mixture (Sigma Cat. No. L5146, Steinheim, Germany) (all but those otherwise specified were obtained from Gibco Invitrogen Corporation products, Grand Island NY, USA).
  • HAl 9 medium - 96 % DMEM/F12 (Biological Industries, Beth Haemek, Israel) supplemented with 1:1000 dilution of the ITS premix (BD Biosciences, Bedford, MA, USA), 2 mM L-glutamine, 2 ng/ml TGF ⁇ 3 (from R&D Systems Minneapolis MN, USA), 4 ng/ml bFGF, 500 ng/ml ascorbic acid (Sigma, Steinheim, Germany), a 1:1000 dilution of a lipid mixture (Sigma Cat. No.
  • Simfronic 68 P5556 from Sigma, Steinheim, Germany, the stock is 10 %, the F-68 in culture is provided at a concentration of 0.1 %) (Sigma, Steinheim, Germany) (all but those otherwise specified were obtained from Gibco Invitrogen Corporation products, Grand Island NY, USA).
  • CMlOOF medium - 85 % DMEM/F12 (Biological Industries, Biet Haemek, Israel) supplemented with 15 % serum replacement (SR), 2 mM L- glutamine, 0.1 mM ⁇ -mercaptoethanol, 1 % non-essential amino acid stock, 4 ng/ml basic fibroblast growth factor (bFGF) and 100 ng/ml IL6RIL6 chimera (SEQ ID NO:31, a kind gift from Prof. Revel M, the Weizmann Inst. Rehovot, Israel) (all but those otherwise specified were obtained from Gibco Invitrogen Corporation products, Grand Island NY, USA).
  • IL6-IL-6 receptor (IL6RIL6) chimera - 85 % Ko-DMEM, supplemented with 15 % serum replacement (SR), 2 mM L-glutamine, 0.1 mM ⁇ - mercaptoethanol, 1 % non-essential amino acid stock, 4 ng/ml bFGF and 50 ng/ml, 100 ng/ml, 200 ng/ml or 300 ng/ml of IL6RIL6 chimera (Chebath J, et al., 1997 and WO 99/02552 to Revel M., et al. SEQ ID NO:31) (all Gibco Invitrogen Corporation products, Grand Island NY, USA). When used with 100 ng/ml of the IL6RIL6 chimera, this medium is also called
  • hESC lines were passaged every four to six days using 1.5 mg/ml type IV collagenase (Worthington biochemical corporation, Lakewood, NJ, USA). Cells were frozen in liquid nitrogen using a freezing solution consisting of 10 % DMSO (Sigma, St Louis MO, USA), 40 % human serum (HyClone, Utah, USA) and 50 % DMEM/F12 (Biological Industries, Beit Haemek, Israel). Derivation of new hESC lines
  • Blastocyst cultivation - Zygotes were donated by couples undergoing pre- implantation genetic diagnosis (PGD) or in vitro fertilization (IVF) at Cornell Medical College, NY, who signed informed consent forms. The couples underwent the traditional IVF procedure after ovarian stimulation with gonadotropins and oocyte retrieval. Zygotes were cultured to the blastocyst stage according to IVF laboratory standard protocol: under oil using specialized C1/C2 media for insemination, growth and blastocyst development (Cornell).
  • hESC lines After digestion of the zona pellucida by Tyrode's acidic solution (Sigma, St Louis MO, USA) or its mechanical removal, the exposed blastocysts were placed in whole on a mitotically inactivated foreskin fibroblasts feeder layer (line F21 which was cultured in an animal free medium since its derivation until used). For the derivation and initial passages, cells were grown in the D2 or HAl 6 culture medium. The cells were initially passaged mechanically every four to ten days. Immunohistochemistry - Undifferentiated hESCs grown in the tested culture system were fixed with 4 % paraformaldehyde and exposed to the primary antibodies (1:50) overnight at 4 °C.
  • SSEA Stage-specific embryonic antigen
  • TRA tumor recognition antigen
  • TRA1-81 tumor recognition antigen
  • Oct 4 Synerg.
  • Karyotype analysis was performed on at least 20 cells from each sample, two samples per test, as previously described [Amit et al, 2003].
  • Karyotypes were analyzed and reported according to the "International System for Human Cytogenetic Nomenclature" (ISCN).
  • EB formation For the formation of EBs, one to three confluent wells were used in a six-well plate (30 cm 2 ). ESCs were " removed from their culture dish using 1 mg/ml type IV collagenase, further broken into small clumps using 1000 ⁇ l Gilson pipette tips, and cultured in suspension in 58 mm petri dishes (Greiner, Frickenhausen, Germany). EBs were grown in differentiation medium consisting of 80 % DMEM/F12 (Biological Industries, Beit Haemek, Israel), supplemented with 20 % FBSd (HyClone, Utah, USA), and 1 mM L-glutamine (Invitrogen Corporation, Grand Island NY, USA).
  • differentiation medium consisting of 80 % DMEM/F12 (Biological Industries, Beit Haemek, Israel), supplemented with 20 % FBSd (HyClone, Utah, USA), and 1 mM L-glutamine (Invitrogen Corporation, Grand Island NY, USA).
  • RTPCR - Total RNA was isolated from hESCs grown for over 10 passages in feeder-free conditions, or from 10 day-old EBs (created from cells grown in the tested culture system for more then 10 passages) using Tri-Reagent (Sigma, St. Louis MO, USA), according to the manufacturer's instructions.
  • cDNA was synthesized from 1 ⁇ g total RNA using MMLV reverse transcriptase RNase H minus (Promega, Madison WI, USA).
  • PCR reaction included denaturation for 5 minutes at 94 °C followed by repeated cycles of 94 0 C for 30 seconds, annealing for 30 seconds at a temperature as specified in Table 1 and extension at 72 °C for 30 seconds.
  • PCR primers and reaction conditions used are described in Table 1 (see Example 1, hereinabove). PCR products were size-fractionated using 2 % agarose gel electrophoresis. DNA markers were used to confirm the size of the resultant fragments.
  • Teratoma formation cells cultured in the offered culture methods for more than 15 passages, were injected into the rear leg muscle of 4-week-old male SCID-beige mice (two mice for each tested culture system). Cell numbers ranged from 5 x 10 6 cells to 10 7 cells per injection. Three to eight to 12 weeks after injection the mice were sacrificed and the resulting teratomas examined histologically.
  • the basic medium, Dl or D2 is a commercial medium design for industrial and clinical proposes for the culture of hybridomas in suspension.
  • the medium is free from animal, serum products and proteins.
  • HAl 6 and HA 19 are based on defined materials only.
  • Dl medium on a feeder layer-free system is capable of maintaining all hESCs features along with high proliferation rate -
  • the hESCs maintained all hESCs features including high proliferation rates.
  • the hESCs demonstrated a relatively high background differentiation rate of 20 % and low proliferation abilities as compared to hESCs cultured at the same feeder layers systems with the D2 HAl 9 or HAl 6 medium.
  • Dl, D2 and HAl 6 media in feeder layer-free are capable of maintaining hESCs in a proliferative, undifferentiated state, with chromosomal stability and pluripotency - Human ESCs grown in the presence of the D 1 , D2 or HAl 6 medium in feeder-layer free conditions were cultured continuously for up to 53, 24 or 10 passages, respectively, while maintaining their ESC features, including undifferentiated proliferation, chromosomal stability (as tested by karyotype analysis, not shown) and pluripotency.
  • the background differentiation rates were found to be less than 10 %, which is similar to the differentiation rates occurring when hESCs are cultured in the traditional culture system based on MEFs as the feeder layer and medium supplemented with serum replacement and 4 ng/ml bFGF [Amit et al, 2000].
  • Examples of undifferentiated colonies cultured with Dl, D2 or HAl 6 medium in feeder-layer free conditions and with the D2 or HA 16 medium with the tested feeder layers are illustrated in Figures 5a-d.
  • the Dl, D2 or HA16 media are capable of maintaining hESCs with normal population doubling - Similar to cells grown on MEFs, cells cultured with D2 or
  • HAl 6 at the different culture methods. At least 20 cells were tested from each sample, 40 cells from each medium combination. All examined cells were found to sustain normal karyotype of 46,XX for cell lines 13 and 14 and 46,XY for cell line 16 (data not shown). Overall, these results suggest that the cells' karyotype remains stable in the tested conditions, similarly to ESCs grown with MEFs using traditional methods (Amit et al, 2000). hESCs cultured with the Dl, D2 or HA16 express typical cell surface
  • hESCs cultured with the Dl, D2 or H ⁇ 16 medium are pluripotent as tested by EBs formation in vitro -
  • the developmental potential of the cells after prolonged culture in the tested culture methods was examined in vitro by the formation of embryoid bodies (EBs).
  • EBs embryoid bodies
  • EBs formed from the hESCs cultured on the Dl, D2 or HAl 6 medium are capable of differentiating into the ectoderm, endoderm and mesoderm cell lineages
  • Rexl, Cx43 and FGF4 (not shown) [Bhattacharya et al, 2004], cells harvested from 10 day-old EBs expressed genes such as albumin and glucagon (endoderm), ⁇ -cardiac actin, ⁇ -globulin and Flkl (mesoderm), and ACl 33 and neurofilament (ectoderm) as demonstrated by RT-PCR analysis (data not shown).
  • hESCs cultured with the Dl, D2 or HA16 medium are pluripotent as tested by teratomas formation in vivo - The cells pluripotency was also tested in vivo by teratomas formation.
  • hESCs differentiated to representative tissues of the three embryonic germ layers including; cartilage, muscle, bone and fat (mesoderm), stratified epithelium, melanin containing epithelium (ectoderm), and kidney like structure (endoderm and mesoderm), and epithelium of endoderm origin (data not shown).
  • Teratomas formation rates of 90 %, and the number of injected cells were identical to those demonstrated by cells cultured using traditional methods (Amit et al, 2000). Altogether, these results demonstrate that hESCs cells subjected to prolonged culture in the tested culture systems demonstrated all hESCs features including; pluripotency, chromosomal stability, expression of specific genes and surface markers and indefinite proliferation as undifferentiated cells.
  • the HA16 and D2 media are suitable for derivation of hESC line on foreskin fibroblast feeder layers in a complete xeno-free system -
  • the medium combinations of the present invention were also tested for the ability to support hESC line derivation. While using the HAl 6 or D2 medium on foreskin fibroblasts as a supportive layer, new hESC lines were successfully derived and maintained for at least 2 passages (in the presence of the D2 medium) or at least 18 passages (in the presence of the HAl 6 medium).
  • the hESC line derived on foreskin in the presence of the HAl 6 culture medium demonstrated stem cells morphology at passage 18 (and the culture is still ongoing), normal XY karyotype and pluripotency as evidenced by the formation of EBs ( Figures 3a-b and data not shown).
  • the growth and success rates were similar to those obtained while using traditional culture methods. Since the used foreskin fibroblasts line, F21, were derived without any animal products, this new hESC lines were derived under complete xeno-free conditions. Although the new hESC lines still need to be tested for additional hESCs features, their morphology and proliferation rates indicate a typical hESCs culture.
  • hESCs were cultured in suspension, as follows. Materials and Experimental Methods
  • ESCs and culture media - ESC cultures and the tested media Dl medium, D2 medium, HA 16 medium, HA 19 medium and HACMlOO medium, which do not contain serum or serum replacement; and the CMlOOF medium which contains serum replacement, were as described in Example 2, hereinabove.
  • Culture in suspension - To examine the possibility of using the TGF ⁇ - containing medium which is devoid of serum, serum replacement and albumin for scalable culture of hESCs in suspension, hESCs were cultured in suspension in 58 mm petri dishes (Greiner, Frickenhausen, Germany) in a cell density of 1.5 x 10 6 to 6 x 10 6 .
  • the HAl 6 medium was supplemented with 0.1 % F68 (Sigma, St.
  • the cells were passage every 5-7 days using either 30-60 minute incubation with 1.5 mg/ml type IV Collagenase (Worthington biochemical corporation, Lakewood, NJ, USA) or 25 minutes incubation with 1.5 mg/ml type IV Collagenase followed by five minutes incubation with 1 mg/ml
  • DMEM DMEM
  • ko-DMEM DMEM/F12
  • MabADCB MabADCB
  • NCTC medium NCTC medium
  • Blastocyst cultivation - Zygotes were donated by couples undergoing PGD or in vitro fertilization (IVF) at Cornell Medical College, NY 5 who signed informed consent forms. The couples underwent the traditional IVF procedure after ovarian stimulation with gonadotropins and oocyte retrieval. Zygotes were cultured to the blastocyst stage according to IVF laboratory standard protocol: under oil using specialized C1/C2 media for insemination, growth and blastocyst development (Cornell).
  • hESC lines in a suspension culture - Following the removal of the zona pellucida using Tyrode's acidic solution (Sigma, St Louis MO, USA), the trophoblast layer is specifically removed either by immunosurgery or mechanically using 27g needles.
  • the exposed ICM is further cultured in suspension culture with a suitable culture medium (e.g., the CMlOOF, HAl 6 or D2) for 4-10 days. Initially, the cells are mechanically split using 27g needles.
  • a suitable culture medium e.g., the CMlOOF, HAl 6 or D2
  • PCR reactions included denaturation for 5 minutes at 94 0 C followed by repeated cycles of 94 °C for 30 seconds, annealing for 30 seconds at an annealing temperature as specified in Table 1, hereinabove and extension at 72 °C for 30 seconds.
  • PCR primers and reaction conditions used are described in Table 1, hereinbelow.
  • PCR products were size-fractionated using 2 % agarose gel electrophoresis. DNA markers were used to confirm the size of the resultant fragments.
  • EBs-differentiation medium consisting of 80 % DMEM/F12 (Biological Industries, Beit Haemek, Israel), supplemented with 20 % FBSd (HyClone, Utah, USA), and 1 niM L-glutamine (Invitrogen Corporation, Grand Island NY, USA).
  • the ESCs were subject to treatment with 1 mg/ml type IV collagenase and further broken into small clumps using 1000 ⁇ l Gilson pipette tips. 10 day-old EBs were harvested for RNA isolation and histological examination.
  • CMlOOF, HA16, Dl, D2 and HA19 media are suitable for culturing hESCs in suspension - hESCs were cultured in suspension using the newly developed TGF ⁇ -containing medium types which are devoid of serum, serum replacement and albumin.
  • TGF ⁇ -containing medium types which are devoid of serum, serum replacement and albumin.
  • the highest passage of hESCs grown in suspension in the tested medium types were 3 passages in the Dl medium, 7 passages in the D2 medium, 10 passages in the HA 19 medium and 17 passages in the CMlOOF medium.
  • AU hESCs exhibited undifferentiated morphology at these passages and can be further cultured in these media and maintain hESCs features.
  • Histological sections of the hESCs clumps formed in the suspension cultures illustrated homogeneous cell population, of round cells with large nucleus ( Figures 9a-g).
  • the cells were plated back on MEFs, they created colonies with typical hESCs morphology ( Figures 9b-e), and if returned to suspension cultures, they continued proliferation as undifferentiated cells (data not shown).
  • hESCs were cultured in a suspension culture in the presence of the serum or serum replacement-free, IL6RIL6-containing HACMlOO medium, the cells were expanded and maintained in the undifferentiated state for at least 1-2 passages (data not shown).
  • ESCs cultured in suspension are capable of forming EBs - When removed from the Dl, D2 or HAl 6 medium and transferred to EBs medium (80 % DMEM/F12 supplemented with 20 % FBSd and 1 mM L-glutamine), the cells formed EBs containing representative tissues of three embryonic germ layers.
  • EBs medium 80 % DMEM/F12 supplemented with 20 % FBSd and 1 mM L-glutamine
  • Rhesus ESCs can be also cultured in the suspension cultures of the present invention - Similar results with Rhesus ESCs (monkey embryonic stem cells, line R366.4, University of Wisconsin, primate center, Thomson lab, Madison, Wisconsin), which are regarded as good candidate for transgenic model to human diseases, were obtained when the Rhesus ESCs were cultured in suspension in the HAl 6, Dl and D2 TGF ⁇ -containing culture media (data not shown).
  • the new TGF ⁇ -containing medium which is devoid of serum, serum replacement and albumin, or the IL6RIL6-containing medium are capable of supporting the undifferentiated culture of hESCs, while maintaining hESCs characteristics, and provide methods for massive culture of these cells for industrial and clinical purposes.
  • hESCs like mouse ES cells, are traditionally cultured with MEFs, which may expose them to animal pathogens.
  • MEFs MEFs
  • the present inventors have demonstrated, for the first time, a defined animal, serum and feeder layer-free culture system for hESCs, based on the use of commercial medium supplemented with either
  • TGF ⁇ 3 or TGF ⁇ ] andbFGF, and human fibronectin matrix as substitute.
  • This medium is designed for massive cultivation of cells in GMP for industrial or clinical purposes.
  • AU medium types of the present invention (with TGF ⁇ 3 or TGFp 1 ), support hESCs culture.
  • the culture medium with the TGF ⁇ isoform 3 was superior of the culture medium with the TGF ⁇ l isoform in terms of less background differentiation.
  • All medium types of the present invention support the culture with feeders as good as with the regular serum containing media. Cells retained the same proliferation rates and the same background differentiation percentages as hESCs cultured with MEFs using traditional culture methods.
  • the medium can also be used for massive suspended culture of undifferentiated hESCs.
  • TGF ⁇ 3 D2 and HAl 6 media
  • TGFp 1 Dl medium
  • the cells' pluripotency was examined in vitro.
  • Cells cultured in the tested culture systems for more than 10 passages formed EBs similar to those created when grown on MEFs [Itskovitz-Eldor et al, 2000].
  • RT-PCR analysis demonstrated that cells within these EBs differentiated into different cell types representative of the three germ layers.
  • hESCs formed teratomas containing a multitude of tissues types (Dl and D2, HAl 6 in. process). The teratoma formation rates were identical to those of cells cultured with. MEFs.
  • the pluripotency of the cells culture continuously in the tested culture methods remained intact.
  • hESCs can be maintained as undifferentiated cells in the proposed defined animal- and serum-free medium combination, without any feeder cells (Dl 3 D2 and HAl 6) or alternatively, with commonly used acceptable * feeder layers (D2 and HAl 6).
  • these media can facilitate hESCs culture for research, industrial and clinical purposes.
  • these novel culture media were found to support suspended culture of undifferentiated hESCs, the first and primary step in developing a massive culture system for their growth and scale-up, a crucial step for any industrial and clinical uses. The mechanism by which hESCs self-maintain is still unclear.
  • TGF ⁇ family members in hESCs renewal [Amit et al, 2004; Ludwig et al, 2006; James et al, 2005; Chen et al, 2006, Valdimarsdottir & Mummery, 2006]. Further complementary research is required to explain the underlying mechanisms of action of TGF ⁇ at the level of signal transduction, and the fact that TGF ⁇ 3 is more potent than TGF ⁇ i .
  • hESCs Future clinical uses of hESCs will require a reproducible, well-defined and xeno-free culture system.
  • the well- defined media demonstrated in the present study are suitable for culturing hESCs and may be advantageous for undertaking research on the mechanisms of ESC self- maintenance, especially of the possible roles of the TGF ⁇ pathway.
  • Other studies using hESCs, such as the research on differentiation pathways and mechanisms, will benefit from the availability of a well-defined and reproducible culture system.
  • the present invention discloses for the first time:
  • a defined, xeno-free, serum, serum replacement or albumin-free system suitable for both culture and derivation of hESCs.
  • TGF ⁇ 3 was never demonstrated to promote self-renewal of stem cells.
  • a culture system that allows hESC culturing in suspension as undifferentiated without a carrier (without substrate adherence).
  • a scalable culture system suitable for developing control bioprocesses in industrial bioreactors.
  • IL-6 Receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34+CD38-/low cells capable of repopulating Severe Combined Immunodeficiency mice. BLOOD 1999; 94: 923-931.

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Abstract

L'invention concerne des milieux de culture exempts de xéno, bien définis, comprenant un isoforme TGF-bêta ou la chimère formée entre IL6 et le récepteur IL6 soluble (IL6RIL6) et capables de conserver des cellules souches et, notamment, des cellules souches embryonnaires humaines, dans un état non différentié. L'invention concerne, en outre, des cultures de cellules comprenant les milieux de culture et les cellules souches et des procédés d'extension et de dérivation des cellules souches embryonnaires dans de tels milieux de culture exempts de xéno et bien définis. De plus, l'invention concerne des procédés permettant de différentier des ESC ou EB formés à partir de ceux-ci, aux fins de production de cellules spécifiques de lignées.
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AU2006286149B2 (en) 2012-09-13
EP1962719A2 (fr) 2008-09-03
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US8476070B2 (en) 2013-07-02
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US20230060616A1 (en) 2023-03-02
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US9404079B2 (en) 2016-08-02
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